Poster Abstracts

Utilizing human neurons to understand neuropsychiatric disordersZhiping Pang
Child Health Institute of New Jersey
Rutgers-Robert Wood Johnson Medical SchoolAbstract: The pathogenesis and etiology of many neuropsychiatric diseases, such as addiction, eating disorders, schizophrenia, autism spectrum disorders (ASDs) and Rett syndrome (RTT), remain an enigma because studies of the human brain in these patients are largely restricted to brain imaging or post-mortem analyses. Cellular analysis, such as characterization of synaptic transmission, is impossible due to the inaccessibility of human neurons from patients. Recent advancement in stem cell biology has made more in-depth analysis possible. However, modeling of human neuropsychiatric diseases is still in its’ infancy. Current technology of generating neurons from iPS cells is difficult, variable and time consuming. Development of better differentiation protocols and reproducibility of results across platforms are pressing questions to be addressed. I will describe three different methodologies in generating human neurons including: 1) direct conversation of human fibroblasts into functional neurons, i.e. induced neuronal (iN) cells; 2) generating dopaminergic neurons from iPS cells using small molecules and using iN technology; and 3) generating neurons from iPS cells via neuroprogenitor cells. Then I will describe characterization of human neurons derived from patients carrying addiction risk genes, specifically the D398N SNP of nicotinic receptor alpha5 subunit, and from RTT patients. It is clear that with “disease-in-a-dish” models using iPS cell-derived neurons, while they may capture cell-intrinsic properties and synaptic deficits of diseased neurons, special precautions need to be taken into account for the enormous variability in the neuronal subtype composition and neural network identities of iPS cell-derived models for neuropsychiatric disorders.

Generation of transgene-free iPSC lines from patients with Parkinson’s diseaseBi, K., Hermanson, S., Lebekken, C., Piekarczyk, M., Reichling, L., Barron, T., Vogel, K., Life Technologies, Madison, WI; Langston, J. W., Schuele, B., The Parkinson’s Institute and Clinical Center, Sunnyvale, CA.Abstract: Patient-derived induced pluripotent stem cells (iPSCs) offer exciting potential in both cell therapy and in vitro disease modeling. Efficient reprogramming of patient somatic cells to iPSCs plays a key role in realization of these potentials. Many reprogramming methods encounter technical challenges to convert adult/disease somatic cells to iPSCs consistently and with high efficiency. Methods that rely on integrating virus or plasmid to reprogram could potentially result in multiple insertions and risk of tumorigenicity. Reprogramming with episomal vectors, mRNAs and miRNAs has low reprogramming efficiency or requires multiple rounds of transfection and with specific types of cells.
Sendai virus is a negative-strand RNA virus that replicates in the cytoplasm of infected cells and does not integrate into the host genome. Recent papers demonstrated that Sendai virus delivering the four Yamanaka factors is a highly efficient method to reprogram normal human foreskin fibroblasts, peripheral blood mononuclear cells and CD34+ cells to generate integration-free iPSCs.
Here, fibroblasts from skin biopsies of four Parkinson’s disease (PD) patients and two age and gender-matched control individuals were efficiently reprogrammed to iPSCs using the Sendai reprogramming method. These iPSCs are transgene-free and karyotypically normal, express known pluripotency markers and are able to differentiate into embryoid bodies with three germ layers. Gene expression profiles clearly distinguished these iPSCs from their parental fibroblasts and clustered them with other iPSCs and H9 line. Given the efficiency, speed and ease with which we were able to reprogram adult disease fibroblasts, we anticipate Sendai reprogramming method being applied to large scale reprogramming of multiple disease lines potentially in an automated fashion.

TaqMan® assays for rapid assessment of genome, epigenome, and quality of integration-free induced pluripotent stem cellsLakshmipathy, U., R. Quintanilla, J. Fergus, A. Fontes, D. Tieberg, Life Technologies, Carlsbad, CA
C. Vaz, V. Tanavde, Bioinformatics Institute, A*STAR, SingaporeAbstract: Pluripotent stem cells such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are commonly identified and characterized based on biomarker expression. Current methods rely on a combination of in vitro and in vivo cellular methods to confirm pluripotency and tri-lineage differentiation potential. As the bottleneck in efficiency of reprogramming is alleviated with faster and better reprogramming systems, there is a need for high throughput characterization methods that allow for rapid confirmation of the quality of the resulting iPSC.
Molecular analysis platforms offer a quantitative, accurate and fast alternative to current methods and have recently been utilized to qualify pluripotent stem cells. Several platforms are available for gene expression analysis varying in content and complexity. To determine the optimal method and minimal set of genes required for definitive characterization of pluripotency, we have utilized high density array, medium and low density TaqMan® qPCR arrays to compare expression pattern of partially reprogrammed clones and fully reprogrammed iPSC in comparison to parental fibroblast and control embryonic stem cells. Results indicate that focused set of genes in low and medium density arrays can recapitulate the information obtained with large scale arrays with distinct clustering of samples based on their pluripotency that correlated with cellular data. Further, this method was used to identify unique gene that was expressed differentially between partially reprogrammed cells and true iPSC clones as well as pluripotent cells and cells randomly differentiated via embryoid body formation.
Additional assays were carried in parallel to assess epigenome signature using TaqMan Array Human MicroRNA Card and TaqMan assays for copy number variation. Comprehensive analysis of the resulting data indicates similarities between the pluripotent clones but also dissect subtle differences that can be further evaluated for their impact on functionality and long-term stability.Acknowledgements: We thank Harrison Leong for building the algorithm and analysis tool. Special thanks to Dr. Robert Horton and Dr. Kevin Vedvik for their role in shaping this project. The gene content for the TaqMan® hPSCScoreCard™ panel and final reference data were generated under research collaboration with Dr. Alex Meissner of Harvard University. We thank Meissner laboratory researchers Veronika Akopian and Dr. Alex Tsankov for their contributions to the project.

Creation of a human cell model of ataxia-telangiectasia for elucidation of epigenetic mechanisms underlying neurological disease symptomsD’Ecclessis, M. (1), Gruenewald, A. (1), Toro-Ramos, A. (1), Swerdel, M.R. (1), Moore, J.C. (2,3), Hart, R.P. (1,3).
1 Department of Cell Biology & Neuroscience, 2 Department of Genetics, 3 Human Genetics Institute of NJ, RUCDR Infinite Biologics, Rutgers University, Piscataway, NJAbstract: Ataxia-telangiectasia (A-T) is a rare, recessive genetic disorder caused by mutations in the ATM gene, which broadly impacts proper functioning of the nervous and immune systems. Mutations in the ATM gene have been shown to result in an Atm protein deficiency that reduces the ability to detect and repair DNA damage. Despite this knowledge, the cellular mechanism behind A-T and its neurological symptoms is less clear. It is hypothesized that epigenetic mechanisms are directly involved in the neurological symptoms of the disease, which include neurodegeneration and loss of neuronal function. We obtained blood samples from patients affected by A-T as well as from their parents who are carriers for the mutation. These blood samples were reprogrammed using Sendai viral vectors (Cytotune™) to create induced pluripotent stem cell (iPSC) lines that are heterozygous for ATM in the carrier line and compound heterozygous for ATM in the affected line since each inherited allele carries a different ATM mutation. By utilizing genome editing tools that include transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs), we are able to manipulate the ATM gene to produce additional human cell models of A-T. Furthermore, the resulting iPSCs can be differentiated into neurons in order to investigate the epigenetic mechanisms involved in the development of neurological A-T symptoms. We created a cell line that is heterozygous for a mutation in the ATM gene by replacing ATM exon 55 with a puromycin-resistance cassette. Exon 55 is significant because it encodes a portion of the functional kinase domain of Atm. The presence of this cassette and the proper location of its insertion have been experimentally verified by PCR. Together, these carrier, affected, and otherwise manipulated cell lines will serve as invaluable tools to better understand the cellular mechanisms behind the role of epigenetics in neurological symptoms of A-T.Acknowledgements: Supported by the A-T Children’s Project. We thank Dr. Howard Lederman of the A-T Clinical Center at Johns Hopkins University for collecting specimens.